Package Insert Dopamine ELISA Assay Kit 96 Wells For Research Use Only (RUO). Not for use in clinical, diagnostic or therapeutic procedures. v. 1.1 Eagle Biosciences, Inc. 20A Northwest Blvd., Suite 112, Nashua, NH 03063 Phone: 617-419-2019 Fax: 617-419-1110
1. Introduction and Principle of the Test The Eagle Biosciences Dopamine ELISA Assay Kit is intended for the quantitative determination of Dopamine in urine or plasma. The Dopamine ELISA Assay Kit is for research use only and not to be used in clinical, therapeutic or diagnostic procedures. Catecholamine is the name of a group of aromatic amines (noradrenaline, adrenaline, dopamine, and their derivatives) which act as hormones and neurotransmitter, respectively. Adrenaline and noradrenaline are formed from dopamine. They act on the cardiac musculature and the metabolism (adrenaline) as well as on the peripheral circulation (noradrenaline) and help the body to cope with acute and chronic stress. An increased production of catecholamines can be found with tumors of the chromaffine system (pheochromocytoma, neuroblastoma, ganglioneuroma). An increased or decreased concentration of the catecholamines can also be found with hypertension, degenerative cardiac diseases, schizophrenia and manic-depressive psychosis. The Dopamine ELISA assay kit provides materials for the quantitative measurement of dopamine in plasma and urine. Dopamine is extracted using a cis-diol-specific affinity gel and acylated to N-acyl-dopamine and then converted enzymatically into N-acyl-3- methoxytyramine. The competitive Dopamine ELISA Assay kit uses the microtiter plate format. Dopamine is bound to the solid phase of the microtiter plate. Acylated dopamine from the sample and solid phase bound dopamine compete for a fixed number of antiserum binding sites. When the system is in equilibrium, free antigen and free antigen-antiserum complexes are removed by washing. The antibody bound to the solid phase dopamine is detected by anti-rabbit IgG / peroxidase. The substrate TMB / peroxidase reaction is monitored at 450 nm. The amount of antibody bound to the solid phase dopamine is inversely proportional to the dopamine concentration of the sample. 2. Precautions The Dopamine ELISA Assay kit is for research use only and not to be used in clinical, therapeutic or diagnostic procedures. Some reagents contain sodium azide as preservative. Avoid skin contact. Material of animal origin used in the preparation of Dopamine ELISA assay kit has been obtained from animals certified as healthy but these materials should be handled as potentially infectious. Some components of this Dopamine ELISA assay kit are containing hazardous reagents. These components are marked with the adequate hazard label. Dopamine ELISA Assay Kit 2/15
3. Storage and Stability On arrival, store the Dopamine ELISA assay kit at 2-8 C. Once opened the kit is stable until its expiry date. For stability of prepared reagents refer to Preparation of Reagents. Do not use components beyond the expiration date shown on the Dopamine ELISA assay kit labels. Do not mix various lots of any Dopamine ELISA assay kit component within an individual assay. 4. Contents of the Kit Reagents for Sample Preparation: 4.1 Extraction Plate EX-PLATE 2 plates 48 wells coated with boronate affinity gel 4.2. Extraction-Buffer EX-BUFF 1 vial 6 ml, ready for use 4.3 HCl HCL 1 vial 21 ml, ready for use 0.025 M HCl 4.4 Standards (1-7) CAL 1-7 7 vials Each 4 ml, ready for use Concentrations: Standard 1 2 3 4 5 6 7 Dopamine (ng/ml) 0 1.5 10 40 160 640 2,560 Dopamine (nmol/l) 0 9.8 65.3 261 1,045 4,179 16,717 For only determination of urine samples: Standard 2 is not required. For only determination of plasma samples: Standard 7 is not required. 4.5 Control 1 & 2 CON 1 & 2 2 vials Each 4 ml, ready for use Concentrations: see q.c. certificate Dopamine ELISA Assay Kit 3/15
4.6 Acylation Reagent ACYL-REAG 1 vial 6 ml, ready for use Contains DMSO and DMF (please note that solvent reacts with many plastic materials including plastic trays; it does not react with normal pipette tips and with glass devices). 4.7 Acylation Buffer ACYL-BUFF 1 vial 20 ml, ready for use 4.8 Enzyme ENZYME 3 vials each 1.7 ml, lyophilized Catechol-O-methyltransferase 4.9 Coenzyme COENZYME 1 vial 1 ml, ready for use (S-adenosyl-L-methionine) 4.10 Enzyme Buffer ENZYME-BUFF 1 vial 3.5 ml, ready for use Reagents for ELISA: 4.11 Dopamine-Antiserum AS-DA 1 vial 5.5 ml, ready for use, rabbit colour coded green 4.12 MT-Strips STRIPS-DA 1 plate 8 wells each, break apart, precoated with: derivatized dopamine (12 strips), colour coded green 4.13 POD Conjugate CONJ 1 vial Each 12 ml, ready for use, Anti-rabbit IgG-POD conjugate/ peroxidase 4.14 Wash Buffer WASH 1 vial 20 ml, concentrate Dilute content with dist. water to 500 ml total volume 4.15 Substrate SUB 1 vial 12 ml TMB solution, ready for use 4.16 Stop Solution STOP 1 vial 12 ml, ready for use. Contains 0.3 M sulphuric acid 4.17 Adhesive Foil FOIL 10 pieces Ready for use Dopamine ELISA Assay Kit 4/15
Additional materials and equipment required but not provided: Pipettes (15, 20, 50, 120, 300, 700 µl) Repeating dispenser for 20, 25, 50, 100, 150, 200, 250 µl und 1 ml Horizontal shaker Microplate washing device Microplate photometer Distilled water 5. Sample Collection and Storage Plasma Urine EDTA plasma samples are required for the Dopamine ELISA kit. Physical and psychological stress usually causes a high increase of the catecholamine concentration. Therefore, it is recommended to let the patient rest for 20 to 30 minutes after the venipuncture and before collecting the blood sample. Hemolytic and especially lipemic samples should not be used for the Dopamine ELISA assay, because false low values will be obtained with such samples. The plasma samples can be stored at 2-8 C up to 6 hours. For a longer period (up to 1 week) the samples should be stored at -20 C. The total volume of urine excreted during a 24-hours period should be collected and mixed in a single bottle containing 10-15 ml of 6 M hydrochloric acid as preservative. Avoid exposure to direct sun light. Determine the total volume and take an aliquot for the measurement. For patients with suspected kidney disorders the creatinine concentration should be tested, too. Urine samples can be stored at -20 C for at least 6 months. Dopamine ELISA Assay Kit 5/15
6. Preparation of Reagents and Samples 6.1. Preparation of Reagents Wash Buffer Dilute the content of the bottle with distilled water to a total volume of 500 ml. For further use the diluted wash buffer must be stored at 2-8 C for a maximum period of 4 weeks. Enzyme Mix NOTE: The enzyme mix has to be prepared freshly prior to the assay (not longer than 10-15 minutes in advance). After use the reagent has to be discarded. Reconstitute the content of one vial labelled ENZYME with 1.7 ml distilled water. Add 0.3 ml COENZYME and 0.7 ml ENZYME-BUFF (total volume: 2.7 ml) and mix thoroughly. The two additional bottles of ENZYME are allowing a second and a third run of the test. If the whole Dopamine ELISA Kit is to be used in one run it is sufficient to prepare one bottle of enzyme mix. 6.2. Preparation of Samples Allow reagents and samples to reach room temperature. Determinations in duplicates are recommended. Each 20 µl of Standards, Control 1 & 2 and urine samples are extracted. Each 300 µl of plasma samples are extracted. 1. Pipette each 20 µl Standard 1-7, 20 µl Control 1 & 2 and each 20 µl Urine Sample into the respective wells of the extraction plate. Add 250 µl of distilled water to these wells to correct for volume. Pipette each 300 µl Plasma Sample into the respective wells (no volume correction required). 2. Pipette each 50 µl Extraction Buffer into all wells. 3. Incubate 60 minutes at room temperature on an orbital shaker (400-600 r/min). Dopamine ELISA Assay Kit 6/15
4. Decant the plate and remove residual liquid by tapping the inverted plate on a paper towel. 5. Pipette each 1 ml Wash Buffer into all wells and incubate for 5 minutes at room temperature on an orbital shaker (slow shaking). 6. Decant the plate and remove residual liquid by tapping the inverted plate on a paper towel. 7. Pipette each 150 µl Acylation Buffer into all wells. 8. Pipette each 50 µl Acylation Reagent into all wells and continue with step 9. immediately. (please note that solvent reacts with many plastic materials including plastic trays; it does not react with normal pipette tips and with glass devices) 9. Incubate the plate for 20 minutes at room temperature on an orbital shaker (400-600 r/min). 10. Decant the plate and remove residual liquid by tapping the inverted plate on a paper towel. 11. Pipette each 1 ml Wash Buffer into all wells and incubate for 5 minutes at room temperature on an orbital shaker (slow shaking). 12. Decant the plate and remove residual liquid by tapping the inverted plate on a paper towel. 13. Repeat the wash steps 11. and 12. 14. Pipette each 150 µl HCl (0.025 M) into all wells. 15. Incubate the plate with adhesive foil for 20 minutes at room temperature on an orbital shaker (400-600 r/min). Caution: Do not decant the supernatant thereafter. Take each 50 µl of the supernatant for the dopamine assay. Dopamine ELISA Assay Kit 7/15
7. Test Procedure ELISA Allow reagents to reach room temperature. Duplicates are recommended. 1. Pipette each 10 µl of freshly prepared Enzyme Mix into all wells (colour code green). 2. Pipette each 50 µl prepared Standards, Controls and Patient Samples into the respective wells (colour coded green). 3. Incubate the plate with adhesive foil for 30 minutes at room temperature (20 25 ºC) on an orbital shaker (400-600 r/min). 4. Pipette each 50 µl Dopamine-Antiserum (colour coded green) into all wells. 5. Cover the plate with adhesive foil, shake for 10 seconds and incubate for 12 20 hours (overnight) at 2-8 C. 6. Discard or aspirate the contents of the wells and wash thoroughly with each 250 µl Wash Buffer. Remove residual liquid by tapping the inverted plate on clean absorbent paper. Repeat the washing procedure 4 times. 7. Pipette each 100 µl POD-Conjugate into all wells. 8. Incubate for 30 minutes at room temperature on an orbital shaker (400-600 r/min). 9. Washing: Repeat wash step 6. 10. Pipette each 100 µl Substrate into all wells. 11. Incubate 25 to 35 minutes at room temperature (20 25 ºC) on an orbital shaker (400-600 r/min). 12. Pipette 100 µl Stop Solution into all wells. 13. Read the optical density at 450 nm (reference wavelength between 570 and 650 nm) in a microplate photometer within 15 minutes. Dopamine ELISA Assay Kit 8/15
8. Calculation of Results On a semilogarithmic graph paper the concentration of the standards (x-axis, logarithmic) are plotted against their corresponding optical density (y-axis, linear). Alternatively, the optical density of each standard and sample can be related to the optical density of the zero standard, expressed as the ratio OD/ODmax, and then plotted on the y-axis. A good fit is provided with 4 Parameter Logistic (alternatively Log-Logit or Cubic Spline). The concentration of the controls and urine samples can be read off the standard curve directly without any further conversion. The read concentrations of dopamine in plasma samples have to be divided by 15 due to the use of 300 µl plasma sample in relation to 20 µl standard. 8.1. Typical Example Below is listed a typical example of a standard curves with the Dopamine ELISA kit: Dopamine ELISA Assay Kit 9/15
9. Assay Characteristics 9.1. Reference Ranges The reference ranges given below should only be taken as a guideline. It is recommended that each laboratory should establish its own normal values. Dopamine Urine Plasma < 600 µg/day < 100 pg/ml 9.2. Sensitivity The lower limit of detection was determined by taking the 2-fold standard deviation of the absorbance of the Zero Reference and reading the corresponding value from the standard curve. Sensitivity (Urine): Sensitivity (Plasma): Dopamine 0.44 ng/ml 29 pg/ml 9.3. Specificity (Cross Reactivity) Structural related components were tested for possible interference with the antiserum against dopamine used in the Dopamine ELISA kit method. Components Cross Reactivity (%) Dopamine-Ab Adrenaline < 0.020 Noradrenaline 0.23 Dopamine 100 Metanephrine < 0.020 Normetanephrine < 0.020 3-Methoxytyramine 0.28 L-Dopa < 0.01 Tyramine 0.011 Tyrosine < 0.01 Homovanillic acid (HVA) < 0.01 Vanillylmandelic acid (VMA) < 0.01 Dopamine ELISA Assay Kit 10/15
9.4. Recovery Increasing amounts of dopamine were added to a urine and to a plasma sample. Each spiked sample was assayed. The analytical recovery was estimated at different concentrations by using the theoretically expected and the actually measured values. Concentrations in ng/ml Urine added measured expected % recovery Plasma added measured expected % recovery 0.00 26.8 0.00 0.14 9.1 30.4 35.9 85 0.32 0.43 0.47 92 20.6 48.5 47.5 102 0.78 0.87 0.93 94 32.4 52.1 59.2 88 1.17 1.14 1.31 87 51.0 88.6 77.9 114 1.54 1.33 1.68 79 98.5 129.4 125.3 103 1.90 1.93 2.05 94 167.5 204.3 194.3 105 3.65 2.99 3.79 79 390.5 377.1 417.4 90 6.65 5.77 6.79 85 mean recovery: 98 mean recovery: 87 9.5. Linearity The linearity of the Dopamine ELISA kit method was investigated using different dilutions of a urine and a plasma sample. Concentrations in ng/ml dilution Urine recalculated value % Recovery dilution measured measured Plasma recalculated value % Recovery Orig. 480.2 Orig. 11.8 1+1 243.7 240.1 102 1+1 5.98 5.90 101 1+2 148.7 160.1 93 1+2 3.97 3.94 101 1+4 108.9 96.0 113 1+4 2.78 2.36 118 1+9 47.1 48.0 98 1+9 1.45 1.18 123 1+14 33.1 32.0 103 1+14 0.89 0.79 114 mean linearity: 102 mean linearity: 111 Dopamine ELISA Assay Kit 11/15
9.6. Reproducibility 9.6.1. Intra-Assay The reproducibility of the Dopamine ELISA kit method was investigated by determining the intra-assay-coefficients of variation (cv) by repeated measurements of two samples with different concentrations. Concentrations in ng/ml sample N mean value sd cv (%) 1 40 25.1 2.84 11.3 2 40 146 11.6 7.9 Dopamine ELISA Assay Kit 12/15
Pipetting Scheme Sample Preparation Standards Controls Urine Plasma Standard 1-7 µl 20 Control 1&2 µl 20 Patient Urine µl 20 Patient Plasma µl 300 Dist. Water µl 250 250 250 Extraction Buffer µl 50 50 50 50 Incubate 60 minutes at RT (shake: 400-600 r/min) Decant plate and remove residual liquid Wash Buffer ml 1 1 1 1 Incubate 5 minutes at RT (slow shaking) Decant plate and remove residual liquid Acylation Buffer µl 150 150 150 150 Acyl. Reagent µl 50 50 50 50 Immediately shake 20 minutes at RT (shake: 400-600 r/min) Decant plate and remove residual liquid Wash Buffer ml 1 1 1 1 Incubate 5 minutes at RT (slow shaking) Decant plate and remove residual liquid Wash Buffer ml 1 1 1 1 Incubate 5 minutes at RT (slow shaking) Decant plate and remove residual liquid HCl µl 150 150 150 150 Incubate 20 minutes with adhesive foil at RT (shake: 400-600 r/min) Caution: Do not decant the supernatant thereafter For the ELISA take each 50 µl for Dopamine Dopamine ELISA Assay Kit 13/15
Pipetting Scheme - ELISA Dopamine (green) Standards Controls Samples Enzyme Mix (Fresh) µl 10 10 10 Standard 1-7 µl 50 Controls 1&2 µl 50 Samples µl 50 Cover with adhesive foil; shake 30 min at room temperature Dopamine Antiserum µl 50 50 50 Cover the plate with adhesive foil Shake for 10 seconds Incubate for 12 20 hours (overnight) at 2-8 C 4 x washing POD-Conjugate µl 100 100 100 Incubate for 30 minutes at room temperature on an orbital shaker 4 x washing Substrate µl 100 100 100 Incubate 25 to 35 minutes at room temperature on an orbital shaker Stop Solution µl 100 100 100 Reading of absorbance at 450 nm Dopamine ELISA Assay Kit 14/15
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